The fluorescent intensity through time of the photobleached spot were exported using the Slidebook software

The fluorescent intensity through time of the photobleached spot were exported using the Slidebook software. source data underlying Figs.?1e, ?e,2a,2a, d, f, ?f,4b,4b, d, ?d,5b,5b, c, ?c,6c,6c, e, f, ?f,7c,7c, ?c,8bCe,8bCe, 9b, c and Supplementary Figs.?4C7c and 8 are provided as a Source Data file. The raw images were deposited to figshare and can be accessed at 10.6084/m9.figshare.8072438. All other data are included in the manuscript and/or supplemental materials are available from the corresponding authors upon reasonable request. Abstract To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables MC-Val-Cit-PAB-tubulysin5a state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics MC-Val-Cit-PAB-tubulysin5a in vivo. and in living cells lacking HA-tagged histone H2B (chimeric anti-HA scFv, green; mCh-H2B, magenta). From left to Rabbit Polyclonal to CAPN9 right, specifies the chimeric scFv that was generated by loop grafting the 12CA5-scFv CDRs onto the 15F11 scaffold. To screen our chimeras, we fused each with the monomeric enhanced GFP (mEGFP) and co-transfected each of the resulting plasmids into U2OS cells, together with a plasmid encoding 4??HA-tagged red fluorescent protein mCherry fused to histone H2B (4??HA-mCh-H2B). If a chimeric scFv binds to the HA epitope in living cells, it should co-localize with the HA-tagged H2B in the nucleus, as shown in Fig.?1b. Live-cell imaging revealed and (sequences in Supplementary Fig.?1) were superior, displaying little to no misfolding and/or aggregation, strong expression, and co-localization with H2B in the nucleus. In contrast, the other three scFvs did not show any co-localization (Fig.?1c, e). Moreover, in control cells lacking HA tags, both and displayed uniform expression (Fig.?1d, e), indicative of free diffusion without non-specific binding. According to our screen, both and function well, although labels HA tags slightly better than (Fig.?1e). We therefore chose the variant for additional characterization, which we herein refer to as the HA frankenbody due to its construction via grafting. Multicolor labeling of HA-tagged proteins in vivo We tested the HA frankenbody in a variety of different settings. First, since the initial screening had been done with a 4??HA tag, we wanted to see if the HA frankenbody could also bind a 1??HA tag. To test this, we constructed two plasmids: 1??HA fused to the C-terminus of H2B-mCherry (H2B-mCh-1??HA) and 1??HA fused to the N-terminus of mCherry-H2B (1??HA-mCh-H2B). In both cases, the HA frankenbody displayed strong nuclear localization (Fig.?2a). Beyond nuclear proteins, we also wanted to test if the HA frankenbody can work well in the cell cytoplasm, another reducing environment that can interfere with disulfide bond formation33. We tested this by creating a target plasmid encoding the cytoplasmic protein -actin fused with a 4??HA-tag and mCherry (4??HA-mCh–actin). When this plasmid was expressed in cells, co-expressed frankenbodies MC-Val-Cit-PAB-tubulysin5a again took on the localization pattern of their targets, now colocalizing with 4??HA-mCh–actin along filamentous actin fibers (Fig.?2b, left). We therefore conclude that both nuclear and cytoplasmic HA-tagged proteins can be labeled by the HA frankenbody in living cells. Open in a separate window Fig. 2 Multicolor labeling of HA-tagged proteins in vivo. a Frankenbody (FB-GFP; green) labels a 1??HA-tagged nuclear protein, histone H2B (magenta), at the N-terminus or C-terminus in living U2OS cells. Left top: 1??HA at C-terminus (H2B-mCh-1??HA, and used it to immunostain fixed cells expressing HA-tagged H2B or -actin. The purified HA frankenbody stained both the HA-tagged nuclear and cytoplasmic proteins with almost no background (Fig.?3a, b). Open in a separate window Fig. 3 Using purified recombinant frankenbody in vitro. Immunostaining in fixed U2OS cells with purified frankenbody (FB-GFP; green) of an HA-tagged (a) nuclear protein, histone H2B (4??HA-mCh-H2B; magenta; a representative cell image of and plasmid was constructed in two steps: (1) a CDR-loop grafted scFv gblock and a H4K20me1 mintbody 15F11 vector38 linearized by EcoRI restriction sites were ligated via Gibson assembly (House prepared master mix); (2) the linker connecting the scFv and EGFP, as well as EGFP, was replaced by a.